Our overall objective is to understand how the formation of differentiated trophoblast phenotypes is orchestrated in trophoblast progenitor cells. Preliminary results show, surprisingly, that invasive trophoblasts, but not syncytiotrophoblasts, derived from a novel rhesus blastocyst-derived trophoblast stem cell line express the forkhead transcription factor, FoxD3, in their nuclei. Examination of early gestation rhesus placental tissue confirms persistence of FoxD3 expression in cytotrophoblasts. FoxD3 is generally considered to be restricted to progenitor cells in the epiblast and trophectoderm (in the mouse) and to developing neural crest cells. Our siRNA studies suggest that FoxD3 expression is required for the formation of trophoblasts with migratory behavior. 17-estradiol alters the intracellular distribution and expression of FoxD3 and induces syncytiotrophoblast formation. Based on these and other preliminary data we hypothesize that trophoblast differentiation and fate specification depends on a critical expression level and subcellular distribution of FoxD3. This project investigates mechanisms that regulate FoxD3 expression in rhesus trophoblasts using rhesus blastocyst-derived trophoblast stem cells, as well as other in vitro trophoblast differentiation systems and a blastocyst implantation model. The first aim quantifies FoxD3 mRNA levels and determines FoxD3 protein levels in the cytoplasm and nucleus during trophoblast differentiation. The second aim characterizes the effects of FoxD3 silencing and overexpression on trophoblast differentiation and invasion. The third aim uses site-directed mutagenesis to determine the role of phosphorylation in regulating FoxD3 intracellular localization and function. Aim 4 will characterize the effects of 172- estradiol on FoxD3 expression and function. The last aim will complement and extend the studies in the other aims by characterizing the role of FoxD3 in trophoblast differentiation using a rhesus blastocyst implantation model. At the completion of these aims we will have characterized mechanisms which regulate the differentiation of invasive trophoblasts from different trophoblast progenitor cell populations. We will also have uncovered a new role for the transcription factor FoxD3. In addition, we will have provided a rigorous characterization of a novel trophoblast stem cell system that should be useful for further mechanistic studies and the development of regenerative techniques. The placenta is essential for fetal development and successful pregnancy. This research investigates how placental cells are formed from stem cells in the rhesus monkey blastocyst. Understanding these processes could lead to strategies for preventing certain disorders of pregnancy such as miscarriage and preeclampsia.